Nucleotides and nucleosides and their purinoceptors have been found to be important mediators in determining pulmonary vascular (PV) tone. Nucleotides are autacoids; that is, they are released locally, metabolized locally by stereoselective nucleotidases, and act on their own local receptors to bring about changes in vascular tone, and neutrophil and platelet function. The effects of nucleotides and nucleosides on PV tone were first described in 1929 by Drury and Szent-Gyorgi when they demonstrated that the nucleoside adenosine produced a fall in arterial pressure and a rise in pulmonary artery pressure in dogs and cats. Drury A N, Szent-Gyorgi A, J. Physiol (Lond) 68:213-237, 1929. Since this discovery, much research has been performed to characterize the role of adenosine and its specific purinoceptors.
Based on pharmacological analysis in isolated systemic vessels, Burnstock originated the purinergic receptor hypothesis. Burnstock G, Handbook of Physiology-The Cardiovascular System II, 2nd Edition, Volume 2, Chapter 19, pp 567-612, 1979. Adenosine-sensitive receptors, referred to as P.sub.1 receptors, were characterized as having an agonist potency in the order of adenosine &gt;AM &gt;ADP&gt;ATP. These receptors were found to act via an adenylate cyclase system and were antagonized by methylxanthines. Since the original classification was made, P.sub.1 receptors have been subdivided into A.sub.1 and A.sub.2 receptors based upon their effect on adenylate cyclase, receptor affinity and radioligand binding.
A.sub.1 receptors inhibit adenylate cyclase activity. High affinity A.sub.1 receptors have been identified in brain, heart, lung, kidney, skin, pancreas, stomach, spinal cord, intestines, vas deferens, liver, spleen, testis, adrenergic nerve terminals, white blood cells and fat cells. These receptors preferentially bind the purine moiety of adenosine and the order of potency of adenosine analogues is R-phenylisopropyladenosine (R-PIA)&gt;cyclohexyladenosine (CHA)&gt;5'-N-ethylcarboxamidoadenosine (NECA)=2-chloroadenosine (2-CA)&gt;S-phenylisopropyladenosine (S-PIA).
A.sub.2 receptors, on the other hand, stimulate adenylate cyclase activity. Low affinity A.sub.2 receptors have been identified in brain, heart, lung, liver, kidney, thymus, spleen, epididymis, vas deferens, adipose tissue, vascular smooth muscle cells, platelets, fibroblasts, lymphocytes, neutrophils and pheochromocytoma cells. They preferentially bind the ribose moiety of adenosine and follow a potency order NECA&gt;2-CA&gt;R-PIA=CHA&gt;S-PIA. A.sub.2 receptors have been identified in coronary arteries and 2-phenylaminoadenosine (CV1808) was second only to NECA as the most potent coronary vasodilator.
In the heart, A.sub.1 adenosine receptors mediate negative inotropic and negative chronotropic effects while A.sub.2 receptors mediate coronary vasodilation. Effects of agonists and antagonists on A.sub.1 and A.sub.2 adenosine receptors in a variety of tissues have been reported by several different investigators. Bamifylline, a selective A.sub.1 adenosine receptor antagonist, has been demonstrated to prevent thromboxane release in the lung, including immunologically sensitized lungs. Berti et al. Pharmacol. Res. 22:143-150, 1990; Berti et al. Arzneum Forsch/Drug Res. 38:40-44, 1988. Costa et al. (U.S. Pat. No. 5,248,678) disclose a method of treating comatose patients to increase arousal and alertness as measured by the Glascow Coma Score by administering effective amounts of an A.sub.1 and/or A.sub.2 adenosine receptor antagonist. Jacobson et al. (U.S. Pat. No. 4,968,672) teach targeting adenosine receptor antagonists to the brain as central stimulants, to the heart as cardiotonics, to the lung as anti-asthmatics, and to the kidney as diuretics. It is disclosed that an A.sub.1 selective antagonist may be preferable as a diuretic since these antagonists do not decrease total blood flow to the kidney. However, some combination of A.sub.1 /A.sub.2 antagonism may be desirable. By selecting appropriate amine congeners to be administered from the family of xanthine congeners taught having a range of A.sub.1 /A.sub.2 selectivity ratios, one may vary the in vivo selectivity. Jacobson et al., U.S. Pat. No. 4,968,672. Badger et al., U.S. Pat. No. 4,772,607, disclose diallyl analogs of xanthine which act as adenosine antagonists displaying an increased affinity for adenosine A.sub.1 receptors in particular. Use of these analogs as CNS stimulant cognition activators, antifibrillatory agents and bronchodilators is taught. In U.S. Pat. No. 4,783,530, Rzeszotarski et al. also disclose a number of xanthine derivatives which are potent adenosine A.sub.1 receptor antagonists for use as bronchodilators and cardiotonics. However, in subsequent publications, U.S. Pat. No. 5,032,593 and U.S. Pat. No. 5,175,290, disclosing related xanthine derivatives, it is suggested that selectivity of these compounds for the adenosine A.sub.1 receptor may not be responsible for their therapeutic effects. In addition, a method of treating cells having a reduced apical Cl-conductance by contacting these cells with a selective adenosine A.sub.1 receptor antagonist has been disclosed as a treatment for cystic fibrosis. Pollard et al., U.S. Pat. No. 5,366,977.
Adenosine attenuates ischemia-reperfusion injury of the heart upon administration prior to ischemia or reperfusion. Ely, S W et al., J. Thorac Cardiovasc Surg 90:549-556, 1985; Olafsson B, et al. Circ 76:1135-1145, 1987; Lasley, R D, et al., Am J Physiol 263:H1460-H1465, 1992; Ely S W, Berne R M, Circ 85:893-904, 1992; Janier, M F, et al., Am J Physiol 264:H163-H170, 1993; Zhao, Z Q, et al. Circ 88:709-719, 1993. Following 90 minutes of ischemia, an intracoronary infusion of adenosine during reperfusion reduced infarct size, improved regional myocardial blood flow and ventricular function, decreased neutrophil infiltration of the ischemic zone of the myocardium and leukocyte plugging of capillaries, and was associated with preservation of endothelial cell structure. Olafsson B et al., Circ 76:1135-1145, 1987. The mechanisms by which adenosine attenuates the injury in the heart following ischemia and reperfusion are not completely understood. However, it has been determined that by acting on A.sub.1 adenosine receptors, adenosine inhibits the release of neurotransmitter substances, produces negative inotropic and chronotropic responses in the heart, attenuates Ca.sup.2+ overload of cells, and increases glycolytic flux. Ely S W, Berne R M, Circ 85:893-904, 1992; Brechler V et al., J Biol Chem 265:16851-16855, 1990. By acting on A.sub.2 adenosine receptors, adenosine produces vasodilation, inhibits oxygen radical release from neutrophils, inhibits platelet aggregation, and decreases edema formation. Ely S W, Berne R M, Circ 85:893-904, 1992; Haselton F R et al., J Appl Physiol 74:1581-1590, 1993. Adenosine also serves as the primary substrate for ATP synthesis by the purine salvage pathway. When administered prior to ischemia, selective A.sub.1 adenosine receptor agonist, R-PIA, has also been reported to attenuate ischemia-reperfusion injury in the heart. Thornton J D, et al., Circ 85:659-665, 1992. In addition, brief episodes of ischemia (approximately 5 to 15 minutes), also referred to as preconditioning ischemia, have been reported to attenuate ischemia-reperfusion injury in the heart. Thornton J D, et al., Circ 85:659-665, 1992; Lui G S, et al., Circ 84:350-356, 1991; Thornton J D, Am J Physiol 265:H504-508, 1993. However, the positive effects of adenosine and preconditioning ischemia were found to be antagonized by a selective A.sub.1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and a nonselective adenosine receptor antagonist 8-(p-sulfophenyl) theophylline (8-SPT), respectively. Lasley, R D, Mentzer, R M, Am J Physiol 263:H1460-H1465, 1992; Thornton JD, Am J Physiol 265:H504-508, 1993; Toombs C F, et al., Circ 86:986-994, 1992.
In contrast to the heart, adenosine has been reported to cause vasoconstriction in the kidney. A.sub.1 receptor stimulation in the kidney was shown to produce primary vasoconstriction of the afferent arteriole and a decrease in glomerular filtration rate. Suzuki, F., et al., J. Med Chem, 35:3066-3075, 1992. Suzuki et al. found selective and potent antagonism of the A.sub.1 adenosine receptor to be important in diuretic and natriuretic activities of the kidney. It has also been suggested that selective A.sub.1 adenosine receptor blockade is more effective in ameliorating acute renal failure than non-selective antagonism of both the A.sub.1 and A.sub.2 receptors. Kellett, R. et al., Br. J. Pharmacol., 98:1066-1074, 1989. However, Knight, R. J., et al., J. Pharm Pharmacol., 45:979-984, 1993, showed that a selective A.sub.1 adenosine antagonist could only provide protection against endotoxin-induced renal dysfunction in the rat in animals receiving a high dose of endotoxin. Coadministration of the A.sub.1 selective adenosine antagonist DPCPX resulted in statistically significant attenuation of the reduction of renal blood flow and inulin clearance in animals receiving a high dose but not a low dose of endotoxin. From these results, Knight et al. concluded that adenosine does not play a major role in the pathophysiology of endotoxemic ARF.
Adenosine has also been reported to act upon adenosine P.sub.1 receptors in the pulmonary vascular bed to induce vasoconstriction and vasodilation. Neely et al. J. Pharmacol. and Exp. Therap., 250(1):170-176, 1989. In addition, ATP-sensitive purinoceptors are present on pulmonary arteries and veins which produce arterial and venular constriction of pulmonary vessels. Liu et al. J. Pharmacol. Exp. Ther. 251:1204-1210, 1989. It is believed that following ischemia and reperfusion, ectonucleotidase activity is reduced. A reduction in 5' nucleotidase activity can result in a decrease in the concentration of adenosine at A.sub.2 adenosine receptors and an increase in ATP at P.sub.2X purinoceptors, resulting in an increase in pulmonary vascular tone and edema formation. Based upon potency profiles of structural analogues for ATP, ATP-sensitive (P.sub.2) purinoceptors have been subclassified into P.sub.2X and P.sub.2Y purinoceptors. With few exceptions, P.sub.2X receptors are located on vascular smooth muscle cells and mediate vasoconstriction and P.sub.2Y receptors are located on endothelial cells and mediate vasodilation. Burnstock, G. and Kennedy, C., Gen. Pharmac. 16:433-440, 1985; Ralevic et al. Br. J. Pharmacol. 103:1108-1113, 1991. P.sub.2X purinoceptors are characterized by an agonist potency profile of .alpha.,.beta.-methylene ATP (.alpha.,.beta.-MeATP)&gt;.beta.,.gamma.-methylene ATP (.beta.,.gamma.-MeATP)&gt;ATP=2-methylthio ATP (2-MeSATP) and are selectively desensitized with .alpha.,.beta.-MeATP. An agonist potency profile for P.sub.2Y purinoceptors is 2-MeSATP&gt;ATP&gt;.alpha.,.beta.-MeATP, .alpha.,.beta., .gamma.-MeATP. P.sub.2Y purinoceptors are antagonized by the P.sub.2Y receptor antagonist reactive blue 2. Hopwood, A. M. and Burnstock, G., E. J. Pharmacol. 136:49-54, 1987; Burnstock, G. and Warland, J. J. I., Br. J. Pharmacol. 90:383-391, 1987; Houston et al. J. Pharmacol. Exp. Ther. 241:501-506, 1987. Diadenosine pentaphosphate and hexaphosphate derivatives mimic the contractile effects of .alpha.,.beta.-MeATP in the bladder and vas deferens and a synthetic .alpha.,.omega.,-adenine dinucleotide, p.sup.1, P.sup.5 diadenosine 5' pentaphosphate (AP.sub.5 A) desensitizes P.sub.2X receptors suggesting these compounds act on P.sub.2X receptors. Hoyle, C. H. V., Gen. Pharmac. 21:827-831, 1990; MacKenzie et al. Br. J. Pharmacol. 94:699-706, 1988; Stone, T. W., E. J. Pharmacol. 75:93-102, 1981; Hoyle, C. H. V., E. J. Pharmacol. 174:115-118, 1989. Pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS) selectively antagonized the nonadrenergic component of the neurogenic-induced and .alpha.,.beta.-MeATP-induced contractions of rabbit vas deferens suggesting it is a selective antagonist of P.sub.2X purinoceptors. Lambrecht et al. E. J. Pharmacol. 217:217-219, 1992. Moreover, with the use of a selective radioligand for P.sub.2X receptors, [.sup.3 H].alpha.,.beta.-MeATP, and autoradiographic techniques, P.sub.2X purinoceptors have been identified in a variety of systemic blood vessels from different species. Bo, X. and Burnstock, G., J. Vas. Res. 30:87-101, 1993.
Further investigations have been undertaken to understand the mechanisms mediating vasoconstrictor responses to adenosine in the lung in the intact-chest, spontaneously breathing cat under conditions of controlled blood flow and constant left atrial pressure. It was found that adenosine induces vasoconstriction in the lung by acting on an adenosine A.sub.1 -"like" receptor. An A.sub.1 selective agonist was approximately 10 to 30 times more potent than adenosine. It was also found that vasoconstriction response was dependent on formation of thromboxane A.sub.2. Neely et al., J. Pharmacol. and Exp. Therap., 258(3):753-761, 1991. It has also been reported that phorbol myristate acetate (PMA)-induced increases in capillary permeability in the isolated blood-perfused dog lung can be blocked by pretreatment with adenosine, which binds the adenosine A.sub.2 receptors. When an A.sub.1 antagonist, DPCPX, was administered to these animals before PMA introduction in the presence of adenosine, this permeability damage was prevented and the pulmonary vascular resistance remained unchanged from controls. Adkins et al., Appl. Physiol., 1993, 74 (3):982-988. Adkins et al. suggest that this finding leads one to postulate that at least portions of the constriction produced with PMA challenge are mediated by activation of A.sub.1 receptors as evidenced by the blocking effect of DPCPX on the PMA-induced resistance increase. However, as acknowledged by Adkins et al., further studies are required as the mechanisms behind PMA-induced lung injury are poorly understood and exogenous adenosine was present in these experiments. Also, the increase in vascular resistance may not play an important role in lung injury following endotoxin, PMA, or ischemia-reperfusion.
Ischemia-reperfusion injury of the lung occurs after lung transplantation, pulmonary thromboembolectomy or cardiopulmonary bypass. Egan T M, et al., Lung transplantation. Curr Probl Surg 26:675-751, 1989; Levinson R M, et al., Am Rev Resp Dis 134:1241-1245, 1986; Kuratani T, et al., J Thorac Cardiovas Surg 103:564-568, 1992. Ischemia-reperfusion injury of the lung also occurs after ischemia and reperfusion of distant organs, for example the intestines. Schmeling D J, et al., Surg 106:195-201, 1989. In the lung, two hours of ischemia followed by three hours of reperfusion produced structural and functional abnormalities that did not occur with ischemia alone. Murata T, et al., Am Rev Resp Dis 146:1048-1053, 1992; Hamvas A, et al., J Appl Physiol 72:621-628, 1992. Neutrophil infiltration, hemorrhage and edema formation occurred only following reperfusion. In conscious, intact-chest, spontaneously breathing rats, two hours of ischemia alone was associated with minimal structural changes. Murata T, et al., Am Rev Resp Dis 146:1048-1053, 1992. However, two hours of ischemia followed by reperfusion was associated with hemorrhagic necrosis of the lung, disrupted alveoli with exudate, destroyed endothelial cells which were detached from internal elastic lamina, and leukocyte accumulation. In isolated, perfused rabbit lungs, 40 minutes of ischemia (when both ventilation and perfusion were discontinued) followed by 55 minutes of reperfusion was associated with electron microscopic alterations of lung tissue, including gaps between endothelial cell tight junctions, gaps between the capillary lumen and interstitial space and edema formation. Zamora C A, et al., J Appl Physiol 74:224-229, 1993. Following ischemia and reperfusion of these rabbit lungs, the rise in pulmonary artery pressure and increase in wet-to-dry lung weight ratios were associated with an increase in thromboxane. These increases were markedly reduced by administration of a thromboxane receptor antagonist, SQ29548, prior to ischemia. Moreover SQ29548 reduced the alterations in endothelial cell gap junctions and interstitial edema formation on electron microscopy.
It has now been found that administration of an effective amount of an A.sub.1 adenosine receptor antagonist prior to ischemia or during or after reperfusion prevents ischemia-reperfusion injury in these organs or related tissues. Administration of an effective amount of a P.sub.2X purinoceptor antagonist prior to ischemia can also prevent tissue injury resulting from ischemia-reperfusion. Compositions comprising an A.sub.1 adenosine receptor antagonist and/or a P.sub.2X purinoceptor antagonist are useful in the prevention and treatment of ischemia-reperfusion injury following organ transplantation, resulting from surgical procedures, following angioplasty or thrombolytic therapy and associated with certain injuries or disease states. These compositions have also been found to be useful in preventing endotoxin-related tissue injury.